Exosomes are small vesicles of endocytic origin that are released from a variety of cell types under normal or pathological conditions. Although originally described in the 1980's, these vesicles have recently garnered significant attention for their ability to stimulate innate and adaptive immune responses in vitro. In this proposal we predict that exosomes serve a unique role in host responses to Mycobacterium tuberculosis (Mtb) infection. Our main hypothesis is that infected dendritic cells (DCs) and macrophages respond to Mtb-infection by releasing exosomes rich in Mtb antigens and PAMPs, thereby broadcasting stimulation of both innate and adaptive immune receptors beyond the site of infection to enhance immunity to Mtb. The overall objective of this proposal is to determine the biochemical and functional characteristics of exosomes released in response to Mtb-infection. In particular, we will focus on CD4+ T cell responses to exosomes since they are poorly characterized in infection models and may be important for the containment of Mtb-infection. We predict that exosomes provide two signals to promote the genesis of protective CD4+ T cell responses to Mtb. 1. Antigenic signal: Exosomes either directly present peptide-MHC-II complexes to T cells or transfer peptide-MHC-II complexes to recipient DCs for antigen presentation. 2. TLR signaling: CD4+ T cells express TLR2 and respond to TLR2 agonists with enhanced proliferative and cytokine responses upon TCR stimulation. We therefore propose that Mtb TLR2 ligands contained within exosomes generate a second signal during antigen presentation to amplify TCR activation. To assess this model, will first evaluate the biogenesis, composition, and MHC class II antigen presentation capacities of exosomes released by infected DCs and macrophages (Aims 1 and 2). We will then evaluate the ability of TLR2 agonists in Mtb-exosomes to stimulate TLR2 on T cells and enhance T cell responses to Mtb antigens (Aim 3). These studies will test a highly novel function for exosomes in the co-stimulation of T cells responses via T cell-expressed TLRs and will advance our limited understanding of exosome functions in adaptive immunity to Mtb. These studies may also serve as a foundation for the future development of exosome-based diagnostics and therapeutics.